目的 克隆温郁金茉莉酸氨基酸合酶关键基因JAR(JA-amino acid synthetase)的全长cDNA序列,并进行生物信息学和表达分析。方法 根据温郁金转录组数据设计特异性引物。以温郁金根茎的cDNA为模板,通过RT-PCR扩增获得JAR全长cDNA序列,利用生物信息学软件预测基因编码蛋白的特征;利用ClustalW和MEGA 6.06软件构建温郁金JAR的系统进化树;构建原核表达载体PET-22B(+)-JAR,纯化重组蛋白JAR5,并进行鉴定。结果 扩增后的基因编码582个氨基酸,全长1749 bp,且具有典型的GH3特征结构域,不具跨膜域,不存在信号肽,属于不稳定蛋白;PCR扩增后得到约1 800 bp大小的片段,经凝胶过滤色谱纯化,最终得到基于蛋白印迹实验(Western blotting)验证正确的66 200大小的重组蛋白。结论 首次克隆并分析了温郁金JAR基因,纯化了JAR5蛋白,为进一步研究温郁金JAR基因蛋白功能研究提供依据。

Objective To clone the full-length cDNA sequence of the key gene JAR (JA-amino acid synthetase) jasmonic acid synthetase from Curcuma wenyujin, and construct a recombinant plasmid for purification and bioinformatics and expression analysis. Methods Specific primers were designed according to the transcriptome data of C. wenyujin. The JAR cDNA sequence was amplified by RT-PCR from the rhizome of C. wenyujin, and the characteristics of the gene encoding protein were predicted by bioinformatics; By cloning the JAR5_2075 sequence from the rhizome cDNA of C. wenyujin, using bioinformatics software to predict the characteristics of the gene encoded protein, using ClustalW and MEGA 6.06 software to construct the phylogenetic tree of JAR; It constructed the prokaryotic expression vector PET-22B (+)- JAR, purifying and recombining protein JAR5, and identified it. Results The amplified gene encoded 582 amino acids, with a total length of 1749 bp, and had a typical GH3 characteristic domain, without transmembrane domain, and no signal peptide. It was an unstable protein; The size of PCR amplification was about 1800 bp, the fragment was purified by gel filtration chromatography, and finally obtained the correct 66 200 recombinant protein based on Western Blot. Conclusion The JAR gene of C. wenyujin was cloned and analyzed for the first time, and the JAR5 protein was purified, which provided a basis for further research on the protein function of the JAR gene of C. wenyujin.

R286.12

大理药业股份有限公司横向课题（KJHX1603）；温莪术规范化生产及基地建设研究（KJHX2008）